Cyclic implant perfusion, cleaning and passivation process and implant produced thereby

ABSTRACT

This invention is a novel method for perfusion of a porous implant which achieves efficient interpenetration of desired factors into and removal of undesirable factors from the pores of the implant, cleaning of the implant, efficient passivation of the implant (inactivation of pathogens, microorganisms, cells, viruses and the like and reduction in antigenicity thereof), and the novel implant produced by such treatment. The process presents a system wherein the rate of pressure cycling, the fact of pressure cycling, and the amplitude of pressure cycling, results in highly cleaned tissues and other implants for implantation. Target decontamination goals for this process include between about a one (1) to twelve (12) log reduction in bacterial contamination, between about a one (1) to fifteen (15) log reduction in enveloped virus contamination, up to about a five (5) log reduction in non-enveloped virus contamination, between about a two (2) to ten (10) fold reduction in endotoxin, maintenance of implant or graft biologic and biomechanical properties, absence of tissue toxicity due to cleaning solutions used, and reduced implant antigenicity.

2.0 BACKGROUND OF THE INVENTION

2.1 Field of the Invention

This invention is a novel method for perfusion of a porous implant whichachieves efficient interpenetration of desired factors into the pores orchannels of the implant, cleaning of the implant, efficient passivationof the implant (inactivation of pathogens, microorganisms, cells,viruses and the like and reduction in antigenicity thereof), and thenovel implant produced by such treatment.

2.2 Description of Known Procedures for Implant Treatment

As used in this disclosure, the term “implant” refers to any materialthe implantation of which into a human or an animal is considered to bebeneficial. Accordingly, the implant may be tissue-derived material,such as bone, skin, and the like, or it may be a metallic or syntheticmaterial having an internal structure that may require cleaning orsterilization. Bearing this definition in mind, it will be apparent thatmany procedures have been described in the art for treatment of implantsto either clean such implant, inactivate contaminating microorganisms orcells that may be present in or on such implant, or to infuse theimplant with desirable factors. This section of the disclosure discussesseveral known methods for achieving one or more of these results, inorder to more clearly and definitively set forth that which has beeninvented, and which is disclosed and claimed as novel and inventive, asdefined by the claims appended hereto.

European Patent Application No. EP 0 424 159 (Osteotech)—“AsepticProcessing of Allograft Bone and Tissue,” (published Apr. 24, 1991,based on a U.S. Priority application filed Oct. 19, 1989), is anextremely general disclosure relating to aseptic processing of allograftbone and tissue. It appears that the intent of this application was toestablish an early priority date in an effort to foreclose the entirefield of aseptic processing of allograft bone and tissue. However, thedisclosure is so general that it does not appear to contain an enablingdisclosure of any protectible allograft sterilization method. No U.S. orEuropean patent related to this extremely general published applicationappears to have ever issued.

In U.S. Pat. No. 5,333,626 (Cryolife)—“Preparation of Bone forTransplantation”, (issued on Aug. 2, 1994, based on an application filedon Dec. 31, 1991), relates to a method of preparing bone fortransplantation by maintaining the internal matrix of the bone to beimplanted, preferably at high pressure, in the presence of adecontaminating agent, preferably polyvinyl pyrrolidine-iodine (PVP-I)optionally in the presence of a detergent, in solution. The “highpressure” feature of this patent is described at column 5, lines 10-31:“High pressure washing conditions should provide a force sufficient todrive the cleaning solution into internal matrix of the bone. Such highpressure washing conditions include, for example, vigorous agitation,such as with a paint can shaker, or high pressure lavage such as with ahigh pressure liquid jet stream . . . The pressure of the liquid jetstream is preferably 100 to 3,000 psi and most preferably 500 to 1,500psi.” However, the patent does not disclose or suggest exposure of animplant to an oscillating atmospheric pressure, the referenced patentrequires pressures significantly higher than those required according tothe present invention, and it is only applicable to bone, while thepresent invention is applicable to bone or soft tissue. In addition, theclaimed process requires approximately 1-2 days to complete.

In U.S. Pat. No. 5,513,662 (Osteotech)—“Preparation of Bone forTransplantation” (this patent issued on May 7, 1996 as acontinuation-in-part of the application that issued as the U.S. Pat. No.5,333,626 patent, based on an application filed on Jan. 21, 1994, andclaiming priority to the Dec. 31, 1991 filing date of the application onwhich the U.S. Pat. No. 5,333,626 patent is based), relates to a methodof preparing bone for transplantation in which the internal matrix ofthe bone is maintained at a pressure below one atmosphere. It isdisclosed (column 10, lines 13-19) that “optimum times for maintainingpressure below ambient are generally in the range of 30 to 60 minutesbut can be determined for each application by monitoring progress ofblood and lipid extraction (see Example 10).” It is further disclosedthat generally use of gas pressure below ambient for less than twominutes will be ineffective and use for longer than five hours willconfer no further benefit. Thus, the '662 patent requires that the bonebe maintained for substantial periods of time at pressures below oneatmosphere. There is no disclosure or suggestion of rapidly cyclingbetween elevated and decreased pressures, even though it is suggestedthat the bone might first be treated at an elevated pressure, followedby a treatment step at a pressure below atmospheric pressure (see, forexample, claim 3, column 15). The present invention discloses a processwherein transient and cyclical exposure of an implant material to agiven pressure achieves the desired result of implant cleaning,perfusion or passivation.

In U.S. Pat. No. 5,556,379 (LifeNet Research Foundation)—“Process forCleaning Large Bone Grafts and Bone Grafts Produced Thereby,” (issued onSep. 17, 1996 based on an application filed on Feb. 27, 1995, andclaiming priority of an earlier, abandoned application, filed Aug. 19,1994), describes the “Allowash™” process. The patent is explicitlydirected to the removal of “bone marrow from the luminal and cancellousbone spaces in large, essentially whole, bone grafts.” (See Summary ofthe Invention). Accordingly, the referenced patent is directed only totreatment of bone, which has to be largely intact. The stated intent inapplying the process to essentially whole bone grafts is to reduce theload of potentially virus carrying bone marrow to facilitate preparationof smaller bone grafts therefrom. The process involves applying a vacuumto the bone graft to draw solution capable of solubilizing bone marrowthrough articulating cartilaginous surfaces and through the intactbone's intramedullary canal or other bone cavity. The patent neitherdiscloses nor suggests a method in which oscillating pressures are usedto clean a bone graft.

U.S. Pat. No. 5,380,826 (Aphios Corporation)—“Supercritical FluidDisruption of and Extraction from Microbial Cells, (issued on Jan. 10,1995, based on an application filed on Sep. 29, 1992), relates to amethod for harvesting intracellular components by exposing cells to anelevated pressure in the presence of a solvent, and then rapidly andsuddenly releasing the pressure to effect disruption of the cells. Thepatent also discloses an apparatus for carrying out this processcontinuously. However, this patent neither discloses nor suggestsapplying the cell disruption method to allograft bone.

U.S. Pat. No. 5,288,462 (Stephen D. Carter)—“Sterilization Apparatus andMethod” (issued on Feb. 22, 1994, based on an application filed on May18, 1992), describes a chamber for receiving a material to be sterilizedby repeatedly subjecting the chamber to elevated pressures, followed bysudden release of the pressure, i.e. “explosive decompression.” Thepatent requires that the chamber be pressurized to at least 1000 psi.The patent neither discloses, suggests, nor claims application of thismethod or chamber to sterilization of bone materials. There is nodisclosure of cleaning solutions used in connection with the describedapparatus that would be effective in sterilizing the matrix of a bone.There is no disclosure that would allow one skilled in the art todetermine, without undue experimentation, that bone could be sterilizedin this apparatus. In addition, there is no disclosure nor suggestionthat an implant could be sterilized without use of such highly elevatedpressures, but merely by oscillation of lower absolute pressures.

U.S. Pat. No. 5,725,579 (Bioland)—“Process for Treating Bone Tissue andcorresponding Implantable Biomaterials”, (issued Mar. 10, 1998, based ona priority French application filed Dec. 21, 1992 and an earlier U.S.priority filing of Dec. 9, 1993), is directed to a method of cleaningbone by exposing the bone to a supercritical fluid. As best as can beunderstood from this patent, this involves exposing bone to carbondioxide at elevated pressures, in order to solubilize lipids.

Tissue sterilization methods known in the art have undesirableattributes. Gamma irradiation, in order to ensure destruction ofpathogens, such as the human immunodeficiency virus (HIV), has to beused at doses that result in tissue destruction (e.g. 3.5 Mrad; see, forexample, Rasmussen, et al., J. Arthroscopic and Related Surgery,10(2):188-197, (1994); Goertzen, et al., British Soc. of Bone and JointSurg., 77:204-211 (1005); Loty, et al., International Orthopaedics,14:237-242, (1990)). Use of ethylene oxide has been found to result inimplants that produce inflammatory responses (Kudryk, et al., J.Biomedical Materials, 26:1477-148, (1992); Thoren, et al., Clin.Orthopaedics, 318:259-263, (1995); Simonian, et al., Clin. Orthopaedics,302:290-296, (1994); Jackson, et al., Am. J. Sports Medicine, 18:1-9,(1990)). Standard chemical solution treatments, while effective insterilizing surfaces with which the solutions are brought into contact,have the major disadvantage of being insufficiently penetrating to reachthe interstices of tissues, where potentially pathogenic organisms mayreside. In view of these shortcomings, there remains a long-felt-needfor an optimized tissue sterilization process, which would incorporatesome or all of the following features: Effective removal or inactivationof a wide range of bacterial and viral pathogens; absence of grafttoxicity; retention of desirable tissue characteristics, such asbiomechanical strength or growth-inducing properties; effectivenessacross a wide range of operating modifications and for a wide variety oftissue types; ability to conclude the process in a final implant tissuecontainer, to ensure sterile packaging and delivery for implantation.

In view of the foregoing review of the known art relating to implanttreatment and sterilization methods, it is believed that the presentinvention provides a long needed improvement in that no absolutetemperatures or pressures are required to achieve efficient implantcleaning, perfusion, or passivation. In addition, the instant methoddoes not require drilling of holes in implant materials or any othermanipulation or modification in order to achieve efficient implantcleaning and sterilization. Furthermore, the present method permits safepooling of donor tissue for implant production at economies of scale,without at the same time diminishing the desirable biological propertiesof the pooled implant materials. The instant process includes a numberof methodologies, the additive effect of which is the production ofhighly cleansed, sterilized (passivated) tissues, which may beimplanted, without causing toxicity to the recipient. Variousembodiments of the method of this invention includes all of the abovelisted features, namely: effective removal or inactivation of a widerange of bacterial and viral pathogens; absence of graft toxicity;retention of desirable tissue characteristics, such as biomechanicalstrength or growth-inducing properties; effectiveness across a widerange of operating modifications and for a wide variety of tissue types;ability to conclude the process in a final implant tissue container, toensure sterile packaging and delivery for implantation.

3.0 SUMMARY OF THE INVENTION

This invention provides a process wherein an oscillation of pressure iscreated in a chamber containing an implant material in the presence ofvarious cleaning solutions (0.5% tri(n-butyl)phosphate, TNBP; hydrogenperoxide and the like). The process essentially comprises the followingsteps, assuming a metallic or synthetic material having an internalmatrix or space, or manually cleaned (debrided) graft material, whichmay or may not have undergone initial machining, is used as the startingmaterial:

-   -   1. Rapidly evacuate the chamber containing the implant, auto        graft, allograft or xenograft material;    -   2. Rapidly backfill the chamber with cleaning solutions—e.g.        H₂O₂/TritonX-100/TNBP/Betadine mixtures;    -   3. Pressurize chamber;    -   4. Rapidly cycle between steps (1) and (3), for between about        1-150 cycles, maintaining a temperature of between about 35-40        degrees centigrade, with optional application of ultrasonic        energy;    -   5. Machine the product as desired if not previously machined;    -   6. Repeat steps (1)-(4) using the same or a different cleaning        compositions, optionally under elevated or reduced temperature;        and    -   7. Optionally perform a surface decontamination step, preferably        in the final packaging, as in exposure to vapor phase H₂O₂ or        like surface decontamination treatments known in the art.

The absolute pressures of the system do not appear to be extremelycritical to achieving deep, penetrating cleaning of the implant or graftmaterials. Rather, it is the rate of pressure cycling, the fact ofcycling, and possibly the amplitude of pressure cycling, that appears tobe critical to the success of this method. Accordingly, the entireprocess may be successfully conducted at pressures above or below oneatmosphere. Evacuation pressures of 25 inches of mercury to the vaporpressure of the solutions in the chamber are adequate. Backfillpressures of between about 40 and 100 PSI are also adequate. Preferably,the entire process is conducted in a chamber which permits forsonication of the contents throughout or at particular stages of theprocess. In addition, preferably, the entire process is conducted in aprogrammable system under computer or programmable logic circuitcontrol, so that manual processing is minimized and reproducibility ofthe process is maximized. Where the processed tissue is a bone implantor any form of allograft or xenograft tissue, election of appropriatesolvents, such as urea (preferably about 6 M), or other chaotropicreagents, (e.g. 4 M guanidine hydrochloride, or the like), has theadditional advantage of producing a processed tissue of even lowerantigenicity than if such treatment were not included. Targetdecontamination goals for this process include:

-   -   Between about a one (1) to twelve (12) log reduction in        bacterial contamination    -   Between about a one (1) to fifteen (15) log reduction in        enveloped virus contamination    -   Up to about a five (5) log reduction in non-enveloped virus        contamination    -   Between about a two (2) to ten (10) fold reduction in endotoxin    -   Maintenance of implant or graft biologic and biomechanical        properties    -   absence of tissue toxicity due to cleaning solutions used    -   reduced implant antigenicity

Accordingly, it is an object of this invention to provide a method forproduction of safe and effective allograft, autograft, xenograft,metallic or synthetic implants in an efficient, economical manner.

It is a further object of this invention to permit safe pooling oftissue donor sources for implant production, while minimizing the riskthat any single contaminated donor will contaminate any other donortissue or any recipients of the pooled tissue processed according to themethod of this invention.

Another object of this invention is to provide a method for cleaning,perfusing or passivating implant materials without at the same timecompromising the desirable biological properties of the starting implantmaterials.

A further object of this invention is to produce implant materials ofreduced antigenicity.

Further objects and advantages of this invention will become apparentfrom a review of the complete disclosure, including the claims whichfollow.

4.0 BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a schematic in which the cyclic perfusion passivationprocess of the invention through seven cycles is shown, while FIG. 1Bshows the cyclic pressure and fluid exposure to implant materialstreated according to the method of this invention.

FIG. 2 shows a schematic of one embodiment of an apparatus that may beemployed to effect the method according to this invention.

FIG. 3 shows a schematic representation of a further embodiment of anapparatus layout for conducting the method according to this invention.

FIG. 4 provides an overall flow-chart of the various stages ofprocessing an implant according to the cyclic perfusion passivationprocess of this invention from donor tissue acquisition through finalsterile product packaging.

FIG. 5 provides one embodiment of a detailed processing containmentlayout for conducting the method according to this invention.

FIG. 6 is a photograph of a whole humerus after being treated accordingto the method of this invention; a post-cleaning coronal section throughthe head of the humerus reveals the cleanliness of the inner bonematrix.

FIG. 7 is a photograph of an intact knee, including proximal tibia,distal femur and patella, along with articulating tendons and ligaments,before treatment according to the method of this invention.

FIG. 8 is a photograph of the intact knee shown in FIG. 7, aftertreatment according to the method of this invention, showing cleanlinessof the implant, and preservation of the articulating tendons andligaments.

FIG. 9 is a photograph of an anterior aspect of a coronal sectionthrough the proximal femur prior to treatment according to the method ofthis invention.

FIG. 10 is a photograph of the posterior aspect of the coronal sectionthrough the proximal femur shown in FIG. 9, after treatment according tothe method of this invention.

FIG. 11 is a photograph of the sections shown in FIGS. 9 and 10,side-by-side, demonstrating the effectiveness of the treatment accordingto this invention for removal of endogenous substances.

FIG. 12 is a photomicrograph of an osteon from cortical bone withoutfluoroisothiocyanate (FITC) fluorescent dye treatment (400×magnification).

FIG. 13 is a photomicrograph of an osteon from cortical bone afterinclusion of FITC in one of the cleaning solutions of this invention,demonstrating deep interpenetration of the dye into the smallest of boneinterstices—bright green areas indicating structures containing FITC,including the large haversian canal (right margin) and smaller satellitelacunae (central area; 400× magnification).

5.0 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the term “passivate” is intended to refer to theelimination of potentially pathogenic organisms and immunogenicsubstances from an implant. Thus, both sterility and reducedantigenicity is intended by this term, although elimination ofbeneficial biological properties of the implant, such as osteogenicproperties (osteoconduction or osteoinduction; bone fusion), naturaltissue functionality, and desirable structural strength of an implantare not intended by this term. The term “passivation” is preferred tothe term “sterilize” because, while sterilization is a goal, that termhas an absolute connotation which can rarely, if ever, be completelyachieved without attendant tissue destruction. In addition, while theimplants produced according to the method of this invention may not becompletely devoid of any antigenicity or pyrogenicity, these undesirableaspects are greatly reduced, and this too is intended by the term“passivation,” as used herein.

The terms “perfused” or “perfusion,” as used herein, are intended toimply efficient interpenetration of cleaning solutions into and throughthe channels and crevices of materials intended for implantation into arecipient.

As used herein, the terms “rapid” or “rapidly” as they are applied tothe process of pressure cycling according to this invention mean timeframes on the order of seconds to minutes, rather than hours or days.

The terms “sonicate” or “sonication” as used herein mean the applicationof sonic or ultrasonic energy via a container of an implant undergoingprocessing according to the method of this invention under conditionsthat permit efficient transfer of the sonic energy to the implant. Thoseskilled in the art are familiar with the process of sonication andconditions whereby sonic energy may be transferred through a fluid to aworkpiece such that efficient cleaning and bacterial or cellulardisruption is achieved, without resulting in gross, ultrastructuraldamage to the workpiece.

This invention provides a novel method for processing implant materialsincluding, but not limited to, metallic implants, synthetic implants,ceramic implants, autograft, allograft or xenograft materials, includingbone and soft tissue. In particular, soft tissue or allograft bonematerials treated according to the method of this invention permit softtissue or debrided allograft, autograft or xenograft bone to bethoroughly cleaned, machined, sterilized, packaged and then implanted ateconomies of scale heretofore not possible. In the past, tissue bankshave attempted, as much as possible, to process tissue from singledonors, without permitting contact between tissue derived from differentdonors. The concern has been that any given donor tissue may contaminateother donor tissue. Due to the extreme value of any donor's tissue, therisk of a large batch of donor tissues being found to be contaminatedhas been considered an unreasonable risk. However, according to themethod of the present invention, even if heavily contaminated donortissue is included in a batch of pooled donor tissue, the resultantgraft material available for implantation is safe for implantation.

Methods for minimizing the risk that donor tissue will be harvested andprocessed by a tissue bank, referred to herein as “donor qualification”,are known in the art. Accordingly, thorough donor screening, and tissuetesting by enzymatic, immunological, biochemical and molecularbiological techniques are applied to minimize the risk that tissuecarrying pathogens (viruses, bacteria, and the like) will be included inthe materials processed and made available for implantation. Testing forcontamination by human immunodeficiency virus, HIV, hepatitis B virus,HBV, hepatitis C virus, HCV, has now become routine in the art. Knownscreening and qualification methods are desirably included as an initialstep preceding processing of the implant material according to thepresent method. Due to the highly efficient implant cleaning, permeationand passivation process encompassed by the instant invention, it isfurther expected that as yet unidentified potentially pathogenicorganisms or organisms for which routine testing has yet to be developedwill, in any event, be removed from implant materials by virtue of theinstant implant treatment process. Redundancy in the level of implantcleaning that is built into the instant pressure cycling permeation andpassivation process ensures inactivation of such organisms while at thesame time permitting efficient implant processing.

For purposes of the following description, allograft bone is referred toas an exemplary tissue that may be processed according to the presentmethod. However, those skilled in the art will recognize that othertissues, including but not limited to autograft bone, xenograft bone,other porous tissues, synthetic porous materials, and various softtissues, may be processed according to the principles defined herein,without departing from the spirit of the invention exemplified herein byreference to allograft bone material.

According to this invention, allograft bone material from qualifieddonors is first treated by various known bioburden reducing methods, asin cleaning by debriding adventitious tissue according to methods knownin the art. Manual dissection may be employed for removal from the bonesurfaces of ligaments, tendons, skin, fat, muscle, loose bone marrow,and any other non-bone tissue. Alternatively, automated orsemi-automated methods known in the art, (see, for example, the methodsdisclosed in U.S. Pat. Nos. 5,333,626; 5,513,662; 5,725,579, and thelike, herein incorporated by reference for this purpose), may beemployed for initial cleaning of the donor bone material.

At this stage of the process, the cleaned allograft materials fromindividual donors may be pooled and further cleaned as described below.Alternatively, the allograft bone may be machined to the final implantdimensions, followed by pooling with a batch of similarly processed,dimensioned implants for further cleaning as described below. Fortracking purposes, while individual donors would have been tracked up tothis stage, upon pooling, a batch number is defined for furthertracking, with records being maintained of all of the donors that havecontributed to the batch. In yet a further alternative, and to ensureredundancy in the level of cleaning and potentially pathogeniccontaminant inactivation, implant materials from individual donors mayfirst be treated as described below, prior to pooling with implantmaterials from different donors. In this event, the implant materialform individual donors may be further cleaned whole or first machined todesired final dimensions.

When applied to bone, subsequent to initial bioburden reduction andsurface cleaning, the method of this invention provides for furtherprocessing whereby bone marrow, blood, proteins, and particulate matteris efficiently removed, such that what remains is essentially amineralized collagen matrix, in which about a 5 to 6 log reduction inany form of viable organisms (viruses, bacteria, amoebae, rickettsia,fungi) is achieved. As described in greater detail below, this isachieved by a process of pressure cycling or oscillation, employing avariety of cleaning and sterilization solutions which are caused toefficiently interpenetrate the matrix. By repeated cycling and changingof the cleaning solvents, the channels of essentially any porous matrixare unclogged, and cleansed. A pre-defined, pre-programmed cycle ofwashes is employed, preferably with concurrent ultrasonic bombardment,to achieve penetrating sterilization of the implant. We have found thatthe combination of oscillating fluid pressure and ultrasonic energyaccelerates solution interpenetration and endogenous substance removal.

In view of the foregoing description, it will be appreciated that in oneembodiment, the invention is a method which comprises the followingsteps:

-   -   1. Rapidly evacuate a chamber containing the implant such as        porous metallic or synthetic materials, autograft, allograft or        xenograft;    -   2. Rapidly backfill the chamber with cleaning solutions—e.g.        H₂O₂/TritonX-100/TNBP/Betadine mixtures;    -   3. Pressurize chamber;    -   4. Rapidly cycle between steps (1) and (3), for between about        1-150 cycles, maintaining a temperature of between about 35-40        degrees centigrade, with optional application of ultrasonic        energy;    -   5. Machine the product as desired if not previously machined;    -   6. Repeat steps (1)-(4) using the same or a different cleaning        compositions, optionally under elevated or reduced temperature;        and    -   7. Optionally perform a surface decontamination step, preferably        in the final packaging, as in exposure to vapor phase H₂O₂ or        like surface decontamination treatments known in the art.

The process of perfusion passivation is further defined with referenceto FIG. 1A. This schematic shows an implant 100 comprising solidstructural constituents 110, channels 120, and adventitious materials130 embedded within the channels 120. The structural constituents 110may be synthetic materials, as in man-made polymeric material, (e.g.poly-L-lactic acid, acrylic acids, and the like), metallic structuralmaterials, or natural materials, such as a mineralized or demineralizedcollagen matrix. The channels 120 may be man-made channels, defined bythe polymerization, molding, melting or other manufacturing process, ormay be natural channels, such as those found in mineralized ordemineralized cancellous or cortical bone matrices. The adventitiousmaterials 130 may be cellular debris, bone marrow, cells, lipids,carbohydrates, proteins, viruses, bacteria, rickettsia, amoebae, fungiand the like. In FIG. 1A, panels (1) and (2) relate to the first stepdescribed above. In panel (1), the channels 120 are primed forback-filling with cleaning solutions by exposing the tissue to decreasedpressures. In panel (2), the cleared channels 120 are shown to besubstantially clear of adventitious materials 130. Panel (3) relates tosteps 2 and 3, wherein molecules of cleaning solution 140 are introducedinto a sealed chamber and are driven into the channels 120 by elevatedpressures. Panel (4) relates to the fourth step described above, whereindecreased pressure removes remaining cellular debris, cleaning solution140, and other remaining adventitious materials from the channels 120,and again primes the matrix for deep penetration, now possible due tothe clarity of the channels 120. In panels (5)-(7), a one cycle repeataccording to the fourth step described above is shown, whereby uponrepressurizing with clean solvents, full interpenetration of thesolvents into the implant matrix is achieved. In panel (6), reducedpressure draws the remaining solution from the implant, which may thenbe dried, as shown in panel (7), prior to further processing (e.g.machining according to step 5 above, further cleaning, according to step6 above), and final packaging of the cleaned tissue. The cycle depictedin FIG. 1A may be repeated as many times as desired to ensure completeinternal cleaning of the matrix interior. In FIG. 1B, a representationof the pressure and fluid oscillation throughout the various steps ofthe above described process is represented.

After being medically released, (i.e. passing a battery of risk factorand biochemical assays, including, for example, HIV-specific PCR, andthe like), donor tissue is cleaned of any extraneous or adventitioustissue. The thus-cleaned tissue is loaded into a sealable reactionchamber. A preferably pre-programmed tissue cleaning process is theninitiated comprising a plurality of wash steps. Deep tissueinterpenetration by cleaning solutions is achieved by oscillating thepressure in the chamber while adding and removing various cleaningsolvents. Ultrasonic energy is applied at various stages of the cleaningprocess to accelerate solution penetration and removal of unwantedcontaminants or endogenous substances, including blood, lipid, andnon-structural or undesired proteins. In one preferred cleaning cycleaccording to this invention, a steps (1-4) of the claimed process arecarried out according to a protocol similar to that defined in thefollowing table to remove blood, fat, bacterial, viral, fungal or othercontamination: TABLE I Duration Step Pressure Fluids* Sonication (min)Purpose 0 Atmospheric None Off NA Load tissue into chamber 1 NegativeNone Off 2 Prime tissue matrix, remove included (60-100 torr) air andloose debris 2 Negative B, C, D, E, On 1 De-gas cleaning fluids (60-100torr) mixtures 3 Positive (5-8 B, C, D, E, On 1 Force fluids into tissuematrix atmospheres) mixtures 4 Negative/ B, C, D, E, On (1 × n) Removedebris loosened by fluids, Positive mixtures pressure oscillation andsonication*Fluids:B = Triton X-100/TNBP, a solvent/detergent to remove debris and killviruses and bacteria;C = 3% hydrogen peroxide, to remove cellular debris, inactivate virusesand bacteria;D = mixture of B and C;E = water-miscible alcohol, such as ethanol or isopropanol;mixtures = B, C, D, E in any desirable proportions.

According to Table I, in step 0, under atmospheric pressure, and nofluid or sonication, a pressurizable chamber in which the process may beconducted, is loaded with metallic, synthetic or other man-made implantmaterials; allograft bone or soft tissue, xenograft bone or soft tissue,from an individual qualified donor. Where the implant is a tissue, thetissue is preferably first cleaned of surface adventitious tissue, priorto initiating the steps shown in table I. In step 1, under negativepressure (vacuum), for a period of about two minutes, the matrix of theimplant or implants is primed (i.e. see FIG. 1, step 1, to removetrapped air, cellular and other loose debris by vacuum). In step 2,under negative pressure, cleaning fluid is introduced with sonication,to aid in penetration of the fluid and to ensure gas is removed from theintroduced fluid. In step 3, under positive pressure, and in thepresence of an appropriate cleaning solvent and sonication, solvent isforced into the matrix of the implant. Thereafter follows a series of“n” cycles of positive and negative pressure in the presence of solventand sonication, during which the matrix channels are backfilled andemptied of solution and debris. The number of times this step is cycledmay be from once to about 150 times (i.e. n=1-150; preferably n is about10-50 times).

After step 4 in Table I, the cleaning fluid is removed to waste underpositive pressure, the tissue is dried under negative pressure, and isrinsed several times under oscillating positive and negative pressureusing sterile water or physiological saline (e.g. phosphate bufferedsaline, PBS), with or without accompanying sonication. The number ofrinse cycles may be from 1-150 times, and is preferably about 1-50times. The rinse solution is drained under positive pressure, and thetissue is again dried under negative pressure.

After removal of the gross contamination according to the steps outlinedabove, the tissue in-process may be machined into dimensionally finishedgrafts if such processing has not previously been accomplished, (step 5of the instant process, as defined above), and then loaded into areaction chamber, same or different than that used to carry out thesteps according to Table I. A deep-penetrating cleaning, passivation orsterilization cycle, preferably under programmable logic control, isthen conducted according to a protocol similar to that defined in TableII (see step 6 defined above, which represent a repeat of steps 1-4 ofTable I, optionally using different cleaning solvents; these steps aredistinguished by indicating the steps as 0′-4′): TABLE II Duration StepPressure Fluids* Sonication (min) Purpose 0′ Atmospheric None Off NALoad tissue into chamber 1′ Negative None Off 2 Prime tissue matrix,remove included (60-100 torr) air and loose debris 2′ Negative F, G, H,I, J, On 1 De-gas cleaning fluids (60-100 torr) mixtures 3′ Positive F,G, H, I, J, On 1 Force fluids into tissue matrix (8-10 mixturesatmospheres) 4′ Negative/ F, G, H, I, J, On (1 × n) Remove debrisloosened by fluids, Positive mixtures pressure oscillation andsonication*Fluids:F = 6 M urea or other chaotropic agents, e.g. 4 M guanidine HCl, toreduce implant antigenicity;G = 1% sodium hypochlorite, to inactivate viruses, bacteria, fungi orother residual contaminants;H = 1 N sodium hydroxide, to inactivate viruses and bacteria;I = 6% hydrogen peroxide, as a sterilant;J = hexane, ether, diethanolamine (DEA), toluene, xylene, butane, CO₂(supercritical), isobutane, propane, acetone, isopropanol, methanol,ketones, ethers, aliphatic or aromatic hydrocarbons, HCl, gasseous HCl.mixtures = F, G, H, I, J in any desirable proportions.

After step 4′ in Table II, the cleaning fluid is preferably retained ina positively pressurized reaction chamber for an extended period toensure complete killing of any residual contaminating pathogens or otherorganisms. A period of from one to sixty minutes, and preferably aboutten minutes, is sufficient for this purpose. The cleaning fluid is thenremoved to waste under positive pressure, the tissue is dried undernegative pressure, and is rinsed several times under oscillatingpositive and negative pressure using sterile water or physiologicalsaline (e.g. phosphate buffered saline, PBS, or the like), with orwithout accompanying sonication. The rinse solution is drained underpositive pressure, and the implant is again dried under negativepressure.

Those skilled in the art will appreciate that the specifics of theprocess outlined according to Tables I and II above may be modified,without departing from the essence of the present invention.Essentially, other cleaning solvents or concentrations than thosesuggested herein may be used, the number of oscillations betweenelevated and reduced pressure, and the cycling times, pressurization anddepressurization levels and periods may be altered, according to therequirements for a given tissue. However, the conditions specified inTables I and II result in deeply penetrating cleaning, as evidenced bythe ability to force dyes deep into tissue matrices, to remove dyes thathave been allowed to soak deep into tissue matrices, and the ability toremove or kill endogenous or added biological contaminants, including awide variety of bacteria, viruses and fungi. Tissues cleaned accordingto this procedure include, but are not limited to: cortical bone,cancellous bone, fascia, whole joints, tendons, ligaments, dura,pericardia, heart valves, veins, neural tissue, submucosal tissue, (e.g.intestinal tissue), and cartilage. Bone treated according to this methodand subsequently tested for retained biomechanical strength and abilityto induce new bone formation (osteoconduction and osteoinduction,collectively referred to as osteogenic activity) retains goodbiomechanical strength and is expected to retain osteogenic activity.Furthermore, bone treated according to one embodiment of this method andimplanted as a xenograft was found to induce little or no adverseimmunological reactivity, indicating reduction in antigenicity of thematerial. This is particularly true where urea or other chaotropicagents (e.g. guanidine hydrochloride), is used as one of the cleaningfluids or is included in a mixture of cleaning fluids.

The method disclosed herein will suggest to those skilled in the art anumber of possible devices to achieve the programmed steps definedabove. Thus, for example, in one embodiment according to this invention,a device such as that shown schematically in FIG. 2 may be employed forsemi-manual implementation of the cyclic perfusion passivation processof this invention. According to this embodiment of the invention, achamber 200 comprising a lid 210 and a trough 220 is adapted for cyclicperfusion passivation of implants. A series of posts 230, onto which aseries of bolts 240 may be tightened are provided for securing the lid210 to the trough 220. A grating 250 is provided inside the chamber 200for receiving implant material to be treated. Through the lid 210 isprovided a series of access ports 260, 261, 262, 263. Access port 260 isa sterile water input line. Access port 261 is an input line for otherfluids. Access port 262 is a vacuum line. Access port 263 is a line forpressure input. in addition, a port 264 is provided for insertion of atemperature probe. Port 265 is a port for supplying power to a sonicatorbuilt into the walls 225 of the chamber 200. Port 266 is a drain.Accordingly, a device such as that shown in FIG. 2 could be usedcarrying out the cyclic perfusion passivation process according to thisinvention.

With reference to FIG. 3, an automated or semi-automated apparatus 300may be defined for carrying out the instant process. Per thisdisclosure, programmable logic controllers activate or deactivate valvesor solenoids 301 a-h at pre-determined times in the cleaning cycle. Animplant is placed in a reaction chamber 310 which is sealed. Anatmospheric vent 320 is provided to permit entrance and removal of wasteand filtered air. Cleaning fluids are introduced into reaction chamber310 from a chemical mixing tank 330 which has a filtered vent toatmosphere 325, to avoid formation of a vacuum in the tank 330. Chemicalfeed lines 340 lead from fluid reservoirs 341 to the chemical mixingtank 330 via a common conduit 345. A programmably controlled pump 350 isoperated to pump appropriately mixed fluids from the tank 330 into thereaction vessel 310. Vacuum or negative pressure is applied to thereaction vessel 310 by means of a vacuum receiver tank 360, in which asource of negative pressure is created by vacuum pump 365. The inclusionof a vacuum reservoir 360 is desirable so that essentially instantaneousvacuum of known dimensions may be applied to the reaction chamber 310,without the need for a vacuum pump such as 365 having to graduallydevelop the negative pressure. Vacuum receiver tank 360 may be evacuatedby pump 365 while reaction tank 310 is under positive pressure. A sourceof sterile water, physiological saline, or like aqueous solution isprovided in storage tank 370, which has a filtered vent 375 to preventformation of a vacuum in tank 370. Pump 376 provides for rapid infusionof aqueous solution into chemical mixing tank 330 for introduction intothe reaction chamber 310. Those skilled in the art will appreciate thatthe water from tank 370 may also be directly introduced into reactiontank 310, without having to first be introduced chemical mixing tank330. Positive pressure is stored in pressure tank 380 which ispressurized by a compressor of filtered gas, to retain sterility in thereaction tank 310. In practice, an appropriately programmed computer orprogrammable logic controllers permit venting of the reaction chamber310, to permit loading of tissue. The chamber is then sealed, evacuated,pressurized, and fluid is introduced and removed, as outlined, forexample, in Table I and Table II above, to complete the implant cleaningprocess.

Manual or automated perfusion of cleaning and sterilizing fluids, asoutlined above, results in reduction of the bioburden of implantmaterial from individual donors, prior to pooling with implant materialsfrom other donors for batch processing. Initial bioburden reduction maybe achieved according to a protocol such as that outlined in Table I, toreduce the potential for contamination of an uncontaminated implant bycontact with a contaminated implant. However, those skilled in the artwill recognize that the penetrating passivation process of thisinvention is so efficient that for certain types of implants in whichthe initial prospect of encountering a contaminated implant issufficiently low, it may be possible to simply batch process implantmaterials according to Table I and Table II, rather than first cleaningimplants from an individual donor according to the Table I program,prior to combining such implant materials from different donors andprocessing the pooled implants according to the Table II program.

Where an initial bioburden reducing step for implant materials derivedfrom individual donors is considered prudent, individual donor tissuesare processed according to the Table I program, and are then quarantineduntil all quality control criteria are passed. Only the individual donortissues that pass such quality control after initial bioburden reductionare pooled for processing according to the Table II protocol. As aninitial bioburden reduction program, a combination of TritonX-100 andTNBP may be used as a first solvent to remove debris and to inactivatebacteria and viruses. A second solvent may be a 3% hydrogen peroxidesolution to remove cellular debris and to further reduce bioburden. Athird solvent may be povidone iodine solution to yet further reducebioburden. Finally, ascorbic acid solution may be employed to decolorizethe implant or remove any residual iodine. These solutions may beemployed in a different order, and indeed, different solutions may beused to similar effect. The particular solutions listed are preferred,however, due to their low toxicity, and our discovery that the definedcombination of solutions results in efficient reduction in bioburden,implant cleaning, passivation and interpenetration. The solutions ofTable I are typically employed in a cycle such as that shown in Table I,steps 0-4.

At this stage of the process, cleaned allograft or xenograft tissue fromindividual donors or previously pooled donors is optionally pooled andfurther cleaned as described below. Alternatively, the tissue is firstdimensioned by machining, trimming and the like, to achieve the finalimplant dimensions. The dimensioned tissue is further processedindividually or is pooled with a batch of similarly or differentlyprocessed, dimensioned implants for further cleaning as described below.For tracking purposes, while individual donors would have been trackedup to this stage, upon pooling, a batch number is defined for furthertracking, with records being maintained of all of the donors that havecontributed to a given batch.

In Table II, a set of solutions is described for achieving penetratingsterilization of individual tissues or tissues pooled from differentdonors which have already been treated according to the program outlinedin Table I. Thus, a first solution of 6% hydrogen peroxide, followed bya second solution of 1% sodium hypochlorite, followed by a solution of 1N sodium hydroxide, may be used to achieve sterilization. A 70% solutionof isopropanol may be used as a broad spectrum germicide. Thus, thesolutions of Table I and Table II may be employed according to theprogram shown, or modified as needed. Those skilled in the art willappreciate that different penetrating sterilants may be employed or thatmixtures of the described sterilants may be possible. In any event, atthe conclusion of this stage of the process, the individual or pooledbatch of implants has been thoroughly cleaned, passivated (if notsterilized), and interpenetrated by cleaning solutions. Reductions inenveloped virus, vegetative bacteria, and fungal contamination of up totwelve logs or higher and of non-enveloped viruses and spores of up toabout five logs are achieved according to the process described herein.In addition, about a two to ten-fold reduction in endotoxin levels isachieved, along with significant elimination of blood, lipid, nucleicacid, and non-structural protein. Furthermore, this process retains thebeneficial structural and other desirable biological properties of theimplant material. Significant enhancements in production yields, throughthe ability to batch process implant from pooled donors, are alsoachieved.

Subsequent to penetrating passivation of the implant materials, theimplant materials are placed in their final packing. Preferably, this isachieved in a sterile environment to avoid introduction of anyadventitious bioburden. To ensure sterile packaging, with the finalmachined grafts in their final, unsealed packages, the implants areexposed to a vapor-phase hydrogen peroxide/peracetic acid or likevapor-phase sterilizing environment. The packages are then closed toensure that no contamination may occur upon removal of the implants fromthe sterile field for storage or shipment to surgeons. The sealedpackages may then, optionally, be subjected to levels of gamma or othertypes of irradiation known to not adversely affect tissue properties(e.g. below about 3.0 Mrad, or for short periods of time to effectsurface sterilization, and to ensure internal destruction of anyresidual large-genome organisms; however, such internal treatment isgenerally not required, deep sterilization having been achievedaccording to the cleaning protocol described herein). Other surface andredundant internal sterilization methods, including exposure to electronbeams, exposure to ethylene oxide, and the like, may also be conductedat this stage, so long as toxicity or diminishment of desirablebiological activities is not thereby effected.

As a further enhancement to the process defined herein is the ability toproduce implant materials with perfusion of desirable bioactivities.Accordingly, in the final rinse steps after steps 0-4 or Table I orsteps 0′-4′ of Table II, a solution containing desired antibiotics,anti-inflammatory drugs or other biologically active agents may beemployed to infuse antibiotic or other desired bioactive substances intothe cleaned, passivated tissues. Alternatively or in addition, growthfactors, such as bone morphogenetic proteins, cartilage derived growthfactors, tissue growth factors, natural or recombinant, and the likeknown in the art may be perfused into the implant.

As can be appreciated from the foregoing detailed disclosure, theprocess of the present invention may be carried out at any stage ofimplant production, and it does not require special preparations such asremoval of cartilage, or potentially implant damaging steps such asdrilling of holes.

As a means of providing an overall concept of the flow of the methodaccording to the present invention, the schematic provided according toFIG. 4 is described. In stage 1, donor materials are introduced into thedonor tissue processing facility and are held in quarantine until thedonor from which the tissue was derived is qualified. In stage 2,released donor materials are surface cleaned by debridement. In stage 3,surface cleaned tissue is machined to produce implants of the desiredfinal dimensions, and are introduced into an automated cyclic perfusionpassivation chamber according to the present invention. In stage 4,implants that have been passivated are introduced into their finalpacking containers and are terminally sterilized by gamma irradiation,vapor-phase exposure to decontaminants, and the like. Finally, in stage5, the passivated and packaged grafts are stored and released afterverification of the sterilization cycles.

In a further embodiment of this invention, a process layout similar tothat shown schematically in FIG. 5 may be employed. According to thislayout, a processing facility 500 shows three parallel and identicaltissue processing facilities A-C. Starting in debridement chambers510A-C, tissue to be treated according to this invention is cleaned anddebrided of gross, adventitious and unwanted tissues. The cleaned tissueis then introduced, via sealable port 515A-C into a reaction chamber310A-C, to which are connected all of the process control andinput/output devices shown in FIG. 3. Upon completion of a cleaningcycle such as that defined according to Table I, tissue is removed viasealable port 516A-C. The cleaned tissue is sorted and stored inquarantine freezers 520A-C, until quality control demonstrates that thetissue is fit for further processing. The released tissues are thentransferred to graft-production rooms 530A-C, where final implantdimensioning and machining is conducted. Following production of thefinally dimensioned implants, the thus processed tissues are loaded intoreaction chambers 310′A-C via sealable port 535. Not shown but connectedto reaction chamber 310′A-C are all the process control and input/outputdevices shown in FIG. 3. Following further cleaning, such as thatdefined according to Table II, the deeply sterilized tissues are removedfrom sealable port 536A-C, and are placed in final packaging. Terminalsterilization is conducted at stations 540A-C, and the terminallysterilized tissues are sealed in the final packaging. The sealedpackages of terminally sterilized tissues are quarantined in freezers545 until final quality control testing permits tissue release tosurgeons.

It will be appreciated that while the process layout provided in FIG. 5is preferred, it is suggestive only, and the process according to theinstant invention may be conducted in other layout formats. Further, itwill be appreciated that according to the layout shown according to FIG.5, it is desirable for the level of ambient particulates to be reducedas tissue is processed through the various stages shown. Thus, while itis adequate for the chamber 510 to be of class 100,000 (100,000particles per billion), it is desirable for areas 520 and 530 to beclass 10,000 or lower. The final packaging area 540 is preferably abouta class 1000 area.

Having generally and in detail described this invention, including itsbest mode, the following specific examples are provided to furtherexemplify, but not to limit, the disclosed invention, the scope of whichshould be reviewed by reference to the claims appended hereto and theequivalents thereof.

EXAMPLES Example 1 Specific Cleaning Protocol for Bone

In one preferred embodiment of this invention, an intact or machinedbone implant is cleaned by treatment sequentially with povidone-I,water, ascorbic acid, TNBP/hydrogen peroxide, water, diethanolamine,water, 6 M urea, water. The sequence of sonication, and pressurefluctuations is conducted according to the sequence defined in Table Ior Table II. It will be appreciated from this disclosure, however, thata wide variety of different cleaning solutions and combinations thereofmay be employed according to the method of this invention. For example,the cleaning solutions may include: sterile water, Triton X-100, TNBP,3% hydrogen peroxide, a water-miscible alcohol, saline solution,povidone iodine, ascorbic acid solution, aromatic or aliphatichydrocarbons, ethers, ketones, amines, urea, guanidine hydrochloride,esters, glycoproteins, proteins, saccharides, enzymes such as proteases(trypsin, pepsin, subtilisin), lipases, sachrases, and the like,gasseous acids or peroxides, and mixtures thereof. The process isconducted at ambient temperatures, elevated temperatures (eighty degreescentigrade) or decreased temperatures. Thus, cleaning of implants in aliquid nitrogen phase (negative eighty degrees centigrade) iscontemplated by this invention.

Example 2 Effectiveness of Process for Implant Cleaning

FIG. 6 is a photograph of a whole humerus after being treated accordingto the method of this invention; a coronal section through the head ofthe humerus reveals the cleanliness of the inner bone matrix.

Example 3 Effectiveness of Process for Cleaning of Hard Tissue and SoftTissue Implants

FIG. 7 is a photograph of an intact knee, including proximal tibia,distal femur and patella, along with articulating tendons and ligaments,before treatment according to the method of this invention.

FIG. 8 is a photograph of the intact knee shown in FIG. 7, aftertreatment according to the method of this invention, showing cleanlinessof the implant, and preservation of the articulating tendons andligaments.

In light of these results, it will be apparent that implant materialsand tissues that may be effectively cleaned according to this procedureinclude, but are not limited to metallic implants, synthetic implants,ceramic implants, allograft, autograft or xenograft tissues. Suchtissues may be selected from tissues comprising: cortical bone,cancellous bone, fascia, whole joints, tendons, ligaments, dura,pericardia, heart valves, veins, neural tissue, submucoal tissue, (e.g.intestinal tissue), and cartilage. Essentially any implantable materialhaving an internal matrix that is required to be cleaned may be treatedto advantage according to the method of this invention.

Example 4 Effectiveness of the Process of this Invention for DeepCleaning of Implants

FIG. 9 is a photograph of an anterior aspect of a coronal sectionthrough the proximal femur prior to treatment according to the method ofthis invention.

FIG. 10 is a photograph of the posterior aspect of the coronal sectionthrough the proximal femur shown in FIG. 9, after treatment according tothe method of this invention.

FIG. 11 is a photograph of the sections shown in FIGS. 9 and 10,side-by-side, demonstrating the effectiveness of the treatment accordingto this invention for removal of endogenous substances and deep,penetrating implant cleaning.

Example 5 Demonstration of the Ability of the Process of this Inventionto Achieve Deep Interpenetration of Cleaning Substances and Impregnationof Implants with Desirable Biologically Active Substances

FIG. 12 is a photomicrograph of an osteon from cortical bone withoutfluoroisothiocyanate (FITC) fluorescent dye treatment (100×magnification).

FIG. 13 is a photomicrograph of an osteon from cortical bone afterinclusion of FITC in one of the cleaning solutions of this invention,demonstrating deep interpenetration of the dye into the smallest of boneinterstices—bright green areas indicating structures containing FITC,including the large haversian canal (right margin) and smaller satellitelacunae (central area; 100× magnification).

These photomicrographs demonstrate that the FITC dye is forced into thesmallest implant interstices, thereby revealing the ability to achievedeep penetrating cleaning. In addition, these photomicrographsdemonstrate that biologically active substances, such as antibiotics,antiviral compounds, anti-inflammatory compounds, growth factors,osteo-inductive substances (e.g. bone morphogenetic protein, cartilagederived morphogenetic protein, natural or recombinant, and the like),when included in solutions employed according to the method of thisinvention, may be effectively imbedded deeply into implant materials.Thus, biologically active substances for permeation into implants,according to the method of this invention are selected from the groupconsisting of bone morphogenetic protein, tissue growth factor beta ormember of the tissue growth factor beta family of growth factors,cartilage derived morphogenetic proteins I or II or both, and anyrelated cartilage derived growth factors, angiogenic factors, plateletderived growth factor. Any of the proteins selected for permeation intoimplants may be natural or recombinant proteins.

1-35. (canceled)
 36. A method for cleaning allograft tissue comprising in any order: i. exposing allograft tissue to a cleaning solution at a pressure greater than atmospheric pressure to cause said cleaning solution to enter the pores of said allograft tissue; ii. exposing said allograft tissue to a pressure less than atmospheric pressure to remove adventitious materials and solution from said pores; and iii. cycling between step (i) and (ii) sufficient times to loosen and remove said adventitious materials in said pores, leaving a cleaned allograft tissue.
 37. The method of claim 36 wherein step (iii) is carried out sufficient times to produce cleaned allograft tissue that is passivated.
 38. The method of claim 37 wherein the allograft tissue produced is suitable for implantation or processing for implantation in humans.
 39. The method of claim 36 wherein said method occurs, at least in part, with concurrent exposure of said allograft tissue to sonication.
 40. The method of claim 36 wherein said cycling is performed in a chamber containing said allograft tissue.
 41. The method of claim 36 wherein said cycling occurs according to a defined program.
 42. The method of claim 36 wherein said cleaning solution is selected from the group consisting of sterile water, Triton X-100, TNBP, 3% hydrogen peroxide, a water miscible alcohol, saline solution, providone iodine, ascorbic acid solution, aromatic hydrocarbons, aliphatic hydrocarbons, ethers, ketone, amines, urea, guanidine hydrochloride, esters, glycoproteins, proteins, saccharides, enzymes, gasseous acids, gasseous peroxides, and mixtures thereof.
 43. The method of claim 36 wherein said cleaning solution is selected from the group consisting of 6% hydrogen peroxide, 1% sodium hypochlorite, 6M urea, 4M guanidine hydrochloride, 1 N sodium hydroxide, isopropanol, water, saline and mixtures thereof.
 44. The method of claim 36 wherein said allograft tissue comprises at least one of the following: cortical bone, cancellous bone, fascia, whole joints, tendons, ligaments, dura, pericardia, heart valves, veins, neural tissue, submucosal tissue, or cartilage, or combinations thereof.
 45. The method of claim 44 wherein said allograft tissue comprises at least one of the following: cortical bone or cancellous bone, or combinations thereof.
 46. The method of claim 44 wherein said allograft tissue comprises at least one of the following: fascia, whole joints, tendons, ligaments, dura, pericardia, heart valves, veins, neural tissue, submucosal tissue, or cartilage, or combinations thereof.
 47. The method of claim 36 wherein said allograft tissue is derived from a single donor.
 48. The method of claim 36 wherein said allograft tissue is derived from a pool of donor tissues.
 49. The method of claim 36, further comprising placing said cleaned allograft tissue into a sterile, sealable package.
 50. The method of claim 49, further comprising performing a surface decontamination step prior to or after sealing said package.
 51. The method of claim 36 wherein the allograft tissue is perfused or coated with a bioactive substance.
 52. The method of claim 51 wherein said bioactive substance is a drug or a growth factor.
 53. The method of claim 52 wherein said growth factor is selected from the group consisting of a bone morphogenetic protein, tissue growth factor beta or member of the tissue growth factor beta family of growth factors, cartilage derived morphogenetic proteins I or 11 or both, and any related cartilage derived growth factors, angiogenic factors, and platelet derived growth factor.
 54. The method of claim 36 wherein step (iii) is carried out sufficient times to produce cleaned allograft tissue having any one or all of the following properties: (a) between about a one to twelve log reduction in bacterial contamination; (b) between about a one to fifteen log reduction in enveloped virus contamination; (c) up to about a five log reduction in non-enveloped virus contamination; (d) between about a two to ten fold reduction in endotoxin; (e) maintenance of implant or graft biologic and biomechanical properties; (f) absence of tissue toxicity due to cleaning solutions used; (g) reduced implant antigenicity.
 55. The method of claim 36 wherein pressures less than atmospheric pressure down to 60 torr are employed.
 56. The method of claim 55 wherein the pressures less than atmospheric pressure are between about 60 to 100 torr.
 57. The method of claim 36 wherein pressures greater than atmospheric pressure up to 10 atmospheres are employed.
 58. The method of claim 57 wherein the pressures greater than atmospheric pressure are between about 6 to 10 atmospheres.
 59. The method of claim 36 wherein said cleaned allograft tissue is pooled with similarly treated tissue, and is further cleaned by conducting steps (i)-(iii) using the same or different cleaning solutions.
 60. The method of claim 36, further comprising the step of: iv. machining said allograft tissue to final dimensions.
 61. The method of claim 60, further comprising the step of: v. conducting steps (i)-(iii) using the same or different cleaning solutions.
 62. The method of claim 60, further comprising placing said cleaned allograft tissue into a sterile, sealable package.
 63. The method of claim 62, further comprising performing a surface decontamination step prior to or after sealing said package.
 64. The method of claim 36 wherein any or all steps are conducted under elevated or reduced temperatures, with respect to ambient temperature.
 65. The method of claim 64 wherein any or all steps are conducted at a temperature between about 37 degrees centigrade and about 80 degrees centigrade.
 66. The method of claim 65 wherein any or all steps are conducted at about 50-60 degrees centigrade.
 67. A method for making an implant which comprises combining allograft tissue to form a composite implant wherein said allograft tissue is treated according to the method of claim
 36. 68. A method for making an implant which comprises combining allograft tissue and other material to form a composite implant wherein at least said allograft tissue is treated according to the method of claim
 36. 69. The method of claim 65 wherein the other material is selected from the group consisting of metallic, synthetic, ceramic, autograft and xenograft materials. 